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Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax , we determined the physiologically significant delayed death effect induced by doxycycline [IC 50(96 h) , 1,401 ± 607 nM]. As expected, IC 50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure., Competing Interests: The authors declare no conflict of interest.

The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need. This study assessed the potential hypnozoiticidal activity of the antibiotic azithromycin, which is thought to exert antimalarial activity by inhibiting prokaryote-like ribosomal translation within the apicoplast, an indispensable organelle. The results show that azithromycin inhibited apicoplast development during liver-stage schizogony in P. vivax and Plasmodium cynomolgi, leading to impaired parasite maturation. More importantly, this study found that azithromycin is likely to impair the hypnozoite's apicoplast, resulting in the loss of this organelle. Subsequently, using a recently developed long-term hepatocyte culture system, this study found that this loss likely induces a delay in the hypnozoite activation rate, and that those parasites that do proceed to schizogony display liver-stage arrest prior to differentiating into hepatic merozoites, thus potentially preventing relapse. Overall, this work provides evidence for the potential use of azithromycin for the radical cure of relapsing malaria, and identifies apicoplast functions as potential drug targets in quiescent hypnozoites., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Human malaria infection begins with a one-time asymptomatic liver stage followed by a cyclic symptomatic blood stage. For decades, the research for novel antimalarials focused on the high-throughput screening of molecules that only targeted the asexual blood stages. In a search for new effective compounds presenting a triple action against erythrocytic and liver stages in addition to the ability to block the transmission of the disease via the mosquito vector, 2-amino-thienopyrimidinone derivatives were synthesized and tested for their antimalarial activity. One molecule, named gamhepathiopine (denoted as "M1" herein), was active at submicromolar concentrations against both erythrocytic (50% effective concentration [EC 50 ] = 0.045 μM) and liver (EC 50 = 0.45 μM) forms of Plasmodium falciparum. Furthermore, gamhepathiopine efficiently blocked the development of the sporogonic cycle in the mosquito vector by inhibiting the exflagellation step. Moreover, M1 was active against artemisinin-resistant forms (EC 50 = 0.227 μM), especially at the quiescent stage. Nevertheless, in mice, M1 showed modest activity due to its rapid metabolization by P450 cytochromes into inactive derivatives, calling for the development of new parent compounds with improved metabolic stability and longer half-lives. These results highlight the thienopyrimidinone scaffold as a novel antiplasmodial chemotype of great interest to search for new drug candidates displaying multistage activity and an original mechanism of action with the potential to be used in combination therapies for malaria elimination in the context of artemisinin resistance. IMPORTANCE This work reports a new chemical structure that (i) displays activity against the human malaria parasite Plasmodium falciparum at 3 stages of the parasitic cycle (blood stage, hepatic stage, and sexual stages), (ii) remains active against parasites that are resistant to the first-line treatment recommended by the World Health Organization (WHO) for the treatment of severe malaria (artemisinins), and (iii) reduces transmission of the parasite to the mosquito vector in a mouse model. This new molecule family could open the way to the conception of novel antimalarial drugs with an original multistage mechanism of action to fight against Plasmodium drug resistance and block interhuman transmission of malaria.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC 50 ], 10.42 μM) and hypnozoites (IC 50 , 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections., (Copyright © 2020 Vanachayangkul et al.)

Background: In the context of malaria elimination/eradication, drugs that are effective against the different developmental stages of the parasite are highly desirable. The oldest synthetic anti-malarial drug, the thiazine dye methylene blue (MB), is known for its activity against Plasmodium blood stages, including gametocytes. The aim of the present study was to investigate a possible effect of MB against malaria parasite liver stages., Methods: MB activity was investigated using both in vitro and in vivo models. In vitro assays consisted of testing MB activity on Plasmodium falciparum, Plasmodium cynomolgi and Plasmodium yoelii parasites in human, simian or murine primary hepatocytes, respectively. MB in vivo activity was evaluated using intravital imaging in BALB/c mice infected with a transgenic bioluminescent P. yoelii parasite line. The transmission-blocking activity of MB was also addressed using mosquitoes fed on MB-treated mice., Results: MB shows no activity on Plasmodium liver stages, including hypnozoites, in vitro in primary hepatocytes. In BALB/c mice, MB has moderate effect on P. yoelii hepatic development but is highly effective against blood stage growth. MB is active against gametocytes and abrogates parasite transmission from mice to mosquitoes., Conclusion: While confirming activity of MB against both sexual and asexual blood stages, the results indicate that MB has only little activity on the development of the hepatic stages of malaria parasites.

A novel 4-aminoquinoline derivative [( S )-7-chloro- N -(4-methyl-1-(4-methylpiperazin-1-yl)pentan-2-yl)-quinolin-4-amine triphosphate] exhibiting curative activity against chloroquine-resistant malaria parasites has been identified for preclinical development as a blood schizonticidal agent. The lead molecule selected after detailed structure-activity relationship (SAR) studies has good solid-state properties and promising activity against in vitro and in vivo experimental malaria models. The in vitro absorption, distribution, metabolism, and excretion (ADME) parameters indicate a favorable drug-like profile., (Copyright © 2017 American Society for Microbiology.)

Primaquine (PQ) remains the sole available drug to prevent relapse of Plasmodium vivax malaria more than 60 years after licensure. While this drug was administered as a racemic mixture, prior studies suggested a pharmacodynamic advantage based on differential antirelapse activity and/or toxicities of its enantiomers. Oral primaquine enantiomers prepared using a novel, easily scalable method were given for 7 days to healthy rhesus macaques in a dose-rising fashion to evaluate their effects on the blood, liver, and kidneys. The enantiomers were then administered to Plasmodium cynomolgi-infected rhesus macaques at doses of 1.3 and 0.6 mg/kg of body weight/day in combination with chloroquine. The (-)-PQ enantiomer had higher clearance and apparent volume of distribution than did (+)-PQ and was more extensively converted to the carboxy metabolite. There is evidence for differential oxidative stress with a concentration-dependent rise in methemoglobin (MetHgb) with increasing doses of (+)-PQ greater than that seen for (-)-PQ. There was a marked, reversible hepatotoxicity in 2 of 3 animals dosed with (-)-PQ at 4.5 mg/kg. (-)-PQ in combination with chloroquine was successful in preventing P. cynomolgi disease relapse at doses of 0.6 and 1.3 mg/kg/day, while 1 of 2 animals receiving (+)-PQ at 0.6 mg/kg/day relapsed. While (-)-PQ was also associated with hepatotoxicity at higher doses as seen previously, this has not been identified as a clinical concern in humans during >60 years of use. Limited evidence for increased MetHgb generation with the (+) form in the rhesus macaque model suggests that it may be possible to improve the therapeutic window for hematologic toxicity in the clinic by separating primaquine into its enantiomers., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Since the 1940s, the large animal model to assess novel causal prophylactic antimalarial agents has been the Plasmodium cynomolgi sporozoite-infected Indian-origin rhesus monkey. In 2009 the model was reassessed with 3 clinical standards: primaquine (PQ), tafenoquine (TQ), and atovaquone-proguanil. Both control monkeys were parasitemic on day 8 post-sporozoite inoculation on day 0. Primaquine at 1.78 mg base/kg/day on days (-1) to 8 protected 1 monkey and delayed parasitemia patency of the other monkey to day 49. Tafenoquine at 6 mg base/kg/day on days (-1) to 1 protected both monkeys. However, atovaquone-proguanil at 10 mg atovaquone/kg/day on days (-1) to 8 did not protect either monkey and delayed patency only to days 18-19. Primaquine and TQ at the employed regimens are proposed as appropriate doses of positive control drugs for the model at present.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.

Long-term in vitro cultures of blood-stage parasites are so far feasible only for Plasmodium falciparum and P. knowlesi. In this chapter, we describe short-term ex vivo culturing of P. cynomolgi and P. vivax. We also describe long-term in vitro culturing of P. knowlesi as well as some techniques for synchronizing parasites. Cultured parasites can be used for a variety of purposes, e.g., for in vitro drug assays and antibody-mediated growth inhibition assays.

There remains a need for new drugs to prevent relapse of Plasmodium vivax or P. ovale infection. The relapsing primate malaria P. cynomolgi has been used for decades to assess drugs for anti-hypnozoite activity. After sporozoite inoculation and blood-stage cure of initial parasitemia with chloroquine, rhesus macaques were treated on subsequent relapses with chloroquine in conjunction with test regimens of approved drugs. Tested drugs were selected for known liver or blood-stage activity and were tested alone or in conjunction with low-dose primaquine. Tinidazole and pyrazinamide prevented relapse when used in conjunction with chloroquine and low-dose primaquine. Triamterene and tinidazole administered without primaquine achieved radical cure in some animals. All other tested drugs or combinations failed to prevent relapse. The rhesus macaque-P. cynomolgi model remains a useful tool for screening drugs with anti-hypnozoite activity. Tinidazole and pyrazinamide require further investigation as agents to enable dose reduction of primaquine.

The antimicrobial and antifungal chemical methylparaben (methyl-4-hydroxybenzoate) was added to the adult sucrose diet of Anopheles gambiae and Anopheles arabiensis, and its effect on longevity was determined. In all cases, significant increases in longevity were observed when 0.2% (w/v) methylparaben was added to meals that were refreshed weekly. When fresh sugar diet was refreshed daily, no increase in longevity was observed due to methylparaben suggesting that the effect of methylparaben is to preserve the quality of the sugar diet. No longevity effect of providing pure water in addition to sugar- or methylparaben-supplemented meals was observed. Feeding preference tests were performed to determine whether meals containing methylparaben were preferred, and whether, when given no choice but the less-preferred diet, mosquitoes would consume less sugar. Using the stable carbon isotope (13)C in paired tests, we show that the sugar diet containing methylparaben was clearly avoided by A. gambiae but not A. arabiensis. Little effect of methylparaben on the total amount of sugar consumed was observed when mosquitoes were given no diet choice. Methylparaben effects on Plasmodium cynomolgi B oocyst formation and encapsulation were observed in a normal A. gambiae stock and one which encapsulates at a high frequency. Nearly two-fold increases in the number of both normal and encapsulated oocysts were observed as a result of methylparaben in the diet. Because of its longevity effects, we have implemented methylparaben use for all mosquitoes in our holdings and recommend it as a routine sugar meal supplement.

A new series of 8-(1-aryl-vinyl)-6,7,10-trioxaspiro [4.5] decanes 7a-e and 3-(1-aryl-vinyl)-l,2,5-trioxaspiro [5.5] undecanes 8a-e have been prepared and screened against multi-drug resistant Plasmodium yoelii in mice. 8-(1-Naphthalen-2-yl-vinyl)-6,7,10-trioxaspiro [4.5] decane 7b, the most active trioxane of the series, has also shown promising activity against Plasmodium cynomolgi in rhesus monkeys.

The gametocytocidal action of a new enamine analogue of primaquine, elubaquine (compound CDRI 80/53, bulaquine), has been evaluated against Plasmodium cynomolgi B in rhesus monkeys. Colony bred Anopheles stephensi mosquitoes were fed on gametocyte carrying rhesus monkeys prior to and at varying intervals after oral administration of a single dose of elubaquine at doses ranging between 0.63 and 5.00 mg/kg. Complete loss of oocyst development and mosquito infectivity was observed within 24 h after administering a single 1.25 mg/kg dose, while higher dose of 3.75 mg/kg inhibited oocyst development within 5 h, indicating gametocytocidal action of the compound. Elubaquine did not show any action against developing oocysts in the vector.

The spectrum of antimalarial activity of the new macrolide antibiotic azithromycin was evaluated against blood- and sporozoite-induced infections with a chloroquine-resistant strain of Plasmodium yoelii nigeriensis (N-67) in Swiss mice and with simian parasite Plasmodium cynomolgi B in rhesus monkeys. Against experimental rodent malaria, a 70 mg/kg/day dose showed curative blood-schizontocidal activity in a four-dose regimen administered orally from day 0 to day 3 or from day 2 to day 5 to mice harboring established infection. The curative response was also obtained with a 40 mg/kg/day dose administered in an extended seven-dose (days 0-6) regimen. Azithromycin was also effective in the causal prophylactic test, since a 50 mg/kg dose from day -1 to day +2 protected mice against P. y. nigeriensis (N-67) sporozoite challenge. In comparison, erythromycin did not show either of the above activities up to a 405 mg/kg/day dose in identical regimens. Comparison of the ED(90) values showed that azithromycin was 31-fold more effective than erythromycin as a blood schizontocide. In the simian model, trophozoite-induced infections of P. cynomolgi B were cured with 25 mg/kg/day azithromycin administered for 7 days. In the causal prophylactic test, the prepatent period was significantly extended in monkeys challenged with P. cynomolgi B sporozoites, presumably because of the growth inhibition of preerythrocytic schizonts in hepatocytes. Azithromycin did not exhibit any hypnozoitocidal (dormant exoerythrocytic stages) activity at 25 mg/kg/day in a seven-dose regimen., (Copyright 2000 Academic Press.)

Arteether (alpha/beta) (a mixture of alpha and beta enantioners) has been reported to possess gametocytocidal activity against Plasmodium cynomolgi B when the drug is given by the intramuscular route, but it would be preferable to use oral route therapy for gametocyte carriers. This is a report of a study of the gametocytocidal action of arteether administered by the oral route. The results indicate high levels of activity at 10 mg/kg in a single dose or in two divided doses when given orally.

The water-soluble artemisinin analogue sodium beta-artelinate, a fast-acting blood schizontocide, was evaluated for gametocytocidal action against simian malaria Plasmodium cynomolgi B, and a single dose of the compound has been found to be an effective gametocytocide by both oral and intravenous routes. The compound was able to sterilize the circulating gametocytes in rhesus monkey, resulting in loss of mosquito infectivity and oocyst development in the Anopheles stephensi. However, no sporontocidal action has been observed with this compound.

Primaquine is the only currently available drug effective against persistent tissue stages of relapsing malaria in humans. Causal prophylactic and radical curative properties of WR182393 (a guanylhydrazone) were investigated as part of an effort to evaluate alternatives to primaquine in the rhesus monkey (Macaca mulatta)/Plasmodium cynomolgi test model. The drug was suspended in dimethylsulfoxide for intramuscular (im) injection. A pilot study indicated causal prophylactic activity in a regimen of 40 mg base/kg/day im for three days beginning the day before intravenous challenge with 1 x 10(6) P. cynomolgi sporozoites. Regimens of 31, 10, 3.1, and 0 mg base/kg/day im for three days were then tested in groups of two monkeys given a similar challenge. The two animals given 31 mg base/kg/day remained parasite-free. Average time to parasitemia for the lower dosage groups was 38, 18, and 8 days respectively. Groups of two monkeys with sporozoite-induced P. cynomolgi infections were also treated for seven days with 31, 10, 3.1, and 0 mg base/kg/day im in combination with 10 mg base/kg/day of chloroquine orally. Both monkeys given 31 mg base/kg/day did not relapse. The average time to relapse following treatment was 48, 29, and 8 days, respectively, for the lower dosage groups. Compound WR182393 is the first non-8-aminoquinoline class of drug to exhibit both causal prophylactic and radical curative properties against a relapsing primate, vivax-like malaria.

The rhesus monkeys (Macaca mulatta) challenged with Plasmodium cynomolgi bastianellii sporozoites were treated with im dexamethasone (Dex) 1 mg.kg-1.d-1 x 7 d and cyclophosphamide (Cyc) 25 mg.kg-1.d-1 x 3d, respectively. On d 7 after challenge, serial sections of liver biopsy stained with HE revealed that the distributive density and size of exo-erythrocytic (EE) form of the parasites showed no difference between medicated and control monkeys. The prepatent period of the primary attack and the relapse patterns after the treatment of Dex and Cyc were also similar to each other. The radically cured monkeys challenged with the Plasmodium erythrocytic form were reinfected with sporozoites, the relapses were much less than those in control monkeys, but the density and size of EE form in liver were similar to those in control monkeys. These results suggest that Dex and Cyc exert no effect on the development of EE form (including hypnozoite) in the monkeys.

The sensitivity of the developing pre-erythrocytic stages of Plasmodium cynomolgi bastianelli to primaquine in single, two or three dose regimens administered at varying times during the incubation period has been investigated. The study reveals that the early pre-erythrocytic stages of the parasite are more susceptible to primaquine than the later stages. Regimens of 5.34 mg/kg x 1 dose on day 0 or day + 1; 2.67 mg/kg x 2 doses on days 0 and 1 and 1.78 mg/kg x 3 doses on days -1, 0, + 1 or days + 1, + 2, + 3 protected all the treated monkeys while identical regimens administered after day 3 of sporozoite inoculation were not curative.

Trifluoroacetyl primaquine oxalate (M8506) was compared with primaquine phosphate for tissue schizontocidal action in rodent and simian malaria. In Plasmodium yoelii sporozoites infected mice, the causal prophylactic effects of M8506 at 5, 10 and 20 mg(base)/kg were 56.7%, 87.2% and 100%, respectively, comparable to those of primaquine (54.4%, 90.8% and 100%). In P. cynomolgi sporozoites infected rhesus monkeys 4 dosage regimens of the two agents were compared for radical curative effect. On the first day of treatment pyronaridine phosphate 10 mg(base)/kg twice a day were intramuscularly injected to eliminate erythrocytic stages of P. cynomolgi. At the dosage of 3.0 mg(base)/kg/day x 3, both M8506 and primaquine radically cured the monkeys. At 0.75 mg/kg/day x 3, 12 of 13 (92.3%) monkeys cured by M8506, 5 of 9 (55.6%) cured by primaquine. At 1.5 and 0.375 mg/kg/day x 3, the radical curative effects of M8506 were also better than those of primaquine. Since the toxicity of M8506 was significantly milder in mice, rats and dogs than that of primaquine, M8506 has potential as a tissue schizontocide.